Polyctenidae (Hemiptera: Cimicoidea) species in the Afrotropical region: Distribution, host specificity, and first insights to their molecular phylogeny

Abstract Polyctenidae bugs are rarely studied, hematophagous, and highly specialized ectoparasites of bats. There are only 32 described species worldwide, including six species in the Afrotropical region. Knowledge on these parasites is limited, and most studies are restricted to the New World polyctenid species. Here we report additional records of Adroctenes horvathi from Kenya and South Africa, as well as Hypoctenes faini from Rwanda. We present an updated list of published polyctenid records in the Afrotropical region indicating their host specificity and their geographical distribution. We report global infection patterns and sex ratio of polyctenids based on previously published data, including Old and New World species. Lastly, we demonstrate the first molecular phylogeny of Polyctenidae, showing their phylogenetic relationship with the closely related family Cimicidae.


| Phylogenetic relation with Cimicidae
The phylogenetic relationship of polyctenids with other groups has previously received little attention. It has been shown that, based on morphological characters, the phylogenetic relationship between cimicids and polyctenids represents two different monophyletic groups, but molecular data were missing from polyctenids (Schuh et al., 2009). Polyctenids are generally excluded from molecular phylogenetic reconstruction of the superfamily Cimicoidea, due to the lack of available specimens and molecular data on these species (Jung & Lee, 2012;Roth et al., 2019;Schuh et al., 2009). Only a cytochrome c oxidase subunit 1 mitochondrial gene (COI) fragment of a North American species, Hesperoctenes fumarius, has been previously published (Smit & Miller, 2019). Additionally, fossil records of polyctenids are not available.

| Reproduction biology of polyctenids
Our knowledge about the basic biology and ecology of these bat bugs is currently based on some long-standing observational work, based on a few common species. The whole life cycle of polyctenids takes place on their hosts (Jordan, 1911;Marshall, 1982a), in contrast with cimicids, which only feed on the host but lay eggs on a substrate, such as the host's roost wall. Polyctenids show strong morphological and physiological adaptation to their parasitic lifestyle; they are viviparous, dorsoventrally flattened, eyeless, and wingless, and these features might strongly affect their host specificity and abundance through limited dispersal ability.

| Host specificity and infection patterns
Previously published data have suggested that polyctenids show a high specificity to their bat hosts. Most species are described as oioxenous (i.e., specific to one certain host species) and/or stenoxenous (i.e., occurring on two or more congeneric host species) (Maa, 1964;Marshall, 1982a). An experimental study has shown that Hesperoctenes fumarius, a New World species, is able to survive and actively feed on different host species, when dispersal barriers are removed (Dick et al., 2009), although congeneric host species were used during this experiment. Overall, specificity and host preferences of polyctenid species are mostly unknown.
Limited data are available about the infection patterns, such as prevalence and abundance of polyctenid species on their hosts.
Hesperoctenes fumarius showed prevalence of 21% on Molossus rufus as well as intensity of infestation (mean number of bat bugs on infected hosts) of 2.22 ± 2.86 (Esbérard et al., 2005).

Presley (2011) also reported the infection patterns of H. fumarius
on two hosts. The prevalence of H. fumarius was 26.8% and 13% on Molossus molossus and M. rufus, respectively. Additionally, he observed mean abundance (mean number of bat bugs per host) of 0.5 ± 1.14 and 0.4 ± 1.49 as well as mean intensity of 2.0 ± 1.43 and 3.2 ± 3.00 on M. molossus and M. rufus, respectively (Presley, 2011).
Hesperoctenes species tend to show sex-biased parasitism toward female bat hosts and in some cases, their abundance is affected by host morphological characters, such as body mass and/or forearm length, which may indicate the body condition of their hosts (Presley & Willig, 2008). Data on the sex ratio of polyctenids are scarce. Some studies reported mostly female biased sex ratio in adults, although sex ratio at emergence was unknown (Maa, 1964;Marshall, 1981Marshall, , 1982a.
Our aim was to describe the specificity, sex ratio, and distributional patterns of polyctenids using published and field collected data along with specimens retrieved from museum collection, extending the current knowledge on the Polyctenidae family. Furthermore, we aimed to gain insights to the phylogenetic relationship of this family in relation to the closely related family Cimicidae, for the first time.

| Sampling and species identification
Opportunistic ectoparasite sampling was carried out by the Centre for Viral Zoonoses at University of Pretoria at several sites in South Africa, Rwanda, and Botswana. This was part of bio surveillance in both frugivorous and insectivorous bat species between 2008 and 2017. Bat species were identified based on morphological characters (Meester, 1986;Van Cakenberghe et al., 2017). Currently valid bat names are used throughout this work, whenever possible, based on batna mes.org (Simmons & Cirranello, 2022). Parasites were individually placed into 70% ethanol. Voucher specimens are deposited at Museum of Zoology, Lausanne, Switzerland. Additionally, further polyctenid specimens were examined at the collection of California Academy of Sciences in San Francisco, CA (USA), and previously unpublished data were also added to this work. Morphological identifications were performed using Maa (1964) and Greenwood (1991).

| DNA extraction and molecular analyses
Polyctenid samples were extracted non-invasively (whole body), keeping whole specimens from external damage. Specimens were placed in separate tubes at 56°C for overnight digestion, using 20 μl Proteinase-K and 180 μl ATL buffer (per sample) (Qiagen). DNA was extracted using DNeasy Blood and Tissue Kits (Qiagen) based on the protocol provided by the manufacturer. We targeted the COI gene (658 bp long fragment) for the molecular analysis, and we used the following primers: Lep1F (5′-ATT CAA CCA ATC ATA AAG ATA TTG G-3′), Lep1Fdeg (5′-ATT CAA CCA ATC ATA AAG ATA TNG G-3′), and Lep3R (5′-TAT ACT TCA GGG TGT CCG AAA AAT CA-3′) (Balvín et al., 2015). Polymerase chain reaction (PCR) master mix was prepared based on previously published protocol (Hornok et al., 2017).
However, the amplification and sequencing of the 28S rRNA gene of Hypoctenes faini were not successful with two different primer sets. PCR reactions of 18S and 28S amplifications were performed as reported (Hornok et al., 2021).
and Microsynth AG performed purification and high-throughput Sanger sequencing of the PCR products.
Sequences (in the following order: 16S rRNA, COI, and 18S rRNA) were concatenated in the Geneious Prime 2019.2.3 (Kearse et al., 2012) software. The alignment of the concatenated sequences was done with MAFFT algorithm (Katoh et al., 2002). The best fitting evolutionary model was selected as general time reversible (GTR) + G + I model by MEGA 11.0.10 (Kumar et al., 2018;Tamura et al., 2021), as it takes into account most parameters. A Bayesian consensus tree was created using the MrBayes (Huelsenbeck & Ronquist, 2001;Ronquist & Huelsenbeck, 2003) Geneious plugin, with GTR model with gamma distribution and invariant sites (GTR + G + I). The stationarity of posterior distribution was also examined using the Geneious plugin. The chain length was set to 5,000,000, sampling frequency to 500 and burn-in length to 100,000. The gene partitions were treated as unlinked. The random seed was set to 21,231. The analysis of the Bayesian tree was done with the MEGA11 11.0.10 (Kumar et al., 2018;Tamura et al., 2021) software. Distribution maps of parasites were produced by using QGIS version 2.16.2.
References sequences of A. horvathi and H. faini can be obtained in GenBank under accession numbers: ON157489-ON182061.

| Geographical distribution of African polyctenids
We collected distributional data of all six African polyctenid species, which have been reported from 14 countries to date (Figure 1a-f, Otomops martiensseni and Rhinolophus simulator, respectively. We excluded records with unspecified data, when exact country was not given (e.g. "Central Africa").

| Infection patterns and sex ratio in Polyctenidae
Published and new records of Polyctenidae prevalence are shown in Table 2, including Old and New World species. Altogether, records of at least 2175 screened host individuals and 1716 parasites were obtained covering broad geographic scale. Most frequently, recorded prevalence rates are known from the New World genus Hesperoctenes. Sex ratio is often female biased in both New and Old World species; however, there is no clear evidence for strong female biased occurrence due to low sampling effort and lack of data.
In total, 645 females and 381 males were reported from previous works, indicating female biased sex ratio ( Table 2). Eoctenes is the most species-rich genus in Africa, with three different species. Nevertheless, E. coleurae seems to be the most rarely collected polyctenid species among all the African Polyctenidae as it has been recorded only once in Sudan and has not been reported since its description (Maa, 1964), making additional conclusions on its distribution problematic. Nevertheless, its host Coleura afra is a widely distributed species, known from several Central, Eastern, and Western African countries. Consequently, E. coleurae might occur within its host distribution (if C. afra is the main host of this species). Future studies focusing on family Emballonuridae and its parasitic fauna should give more insights to the distribution of E.

coleurae.
Eoctenes nycteridis is also endemic to the African continent and has been mostly reported from the central countries with some additional records, such as Eritrea and Liberia; therefore, it is expected to occur in other regions within the distribution range of its hosts, family Nycteridae. Species belonging to family Nycteridae occur in Africa but some parts of Asia as well.

Uganda
Jordan (1912), Maa (1964) Hypoctenes clarus (Jordan, 1922)  and Kenya (Benoit, 1958;Jordan, 1922;Maa, 1970;Patterson et al., 2018). Hypoctenes faini is also a rarely observed species, with only two published records, representing two specimens (Benoit, 1958;Greenwood, 1991). During our work, two specimens of H. faini have been found in Rwanda for the second time ( Figure 1). It might be expected from additional countries where its potential hosts from the Molossidae family are present. Otomops martiensseni, which we recorded in Rwanda as host species, occurs mainly in Central Africa but has populations in the southern and western part of the continent; therefore, the occurrence of H. faini is possible in these areas.

| Host specificity
Based on literature and field collected data, all polyctenid species appeared to be oligoxenous, meaning that they occur on two or more congeneric host species. However, the number of sampled individuals is low and conclusions cannot be drawn on the preferred host species, if any. Nevertheless, all polyctenid species exclusively occur on the members of a single bat family. The level of dispersal ability of polyctenids is unknown, although Marshall (1981) stated that biased sex ratio occurs in polyctenids due to males being the more mobile sex (Marshall, 1981), which could affect their dispersal ability and their specificity. Phylogenetic specificity (rather than ecological specificity) is supported by the fact that some host species

| Sex ratio and infection patterns
Biased sex ratio in ectoparasitic insects is common and has been explored in the case of bat-associated parasites Dittmar et al., 2011;Szentiványi et al., 2017). Several factors may cause biased sex ratio, such as difference between body size, mobility, dispersal ability between sexes, or the presence of reproduction manipulating bacteria or inbreeding Duron et al., 2008;Patterson et al., 2008;Szentiványi et al., 2017).
We found some evidence of female biased sex ratio in polyctenid bat bugs, similarly to previous suggestion (Marshall, 1982a). LMC implies a female biased sex ratio, since males compete for mating opportunities, and mothers try to decrease sexual competition by maximizing female success through reducing the number of male offspring (Hamilton, 1967). Marshall (1982aMarshall ( , 1982b suggested that biased sex ratio occurs because males are more active than females and therefore more exposed to predation by their hosts (Marshall, 1981(Marshall, , 1982a. Additionally, if there is different mobility and dispersal ability between sexes, it might also affect the capture success and thus implies a apparent bias in sex ratio. Furthermore, different longevity between females and males might also strongly influence sex ratio. Dispersal ability and mobility differences between female and male polyctenids are currently unknown on their hosts; however, off-host both sexes are incapable of moving (Marshall, 1982a). Additionally, Wolbachia, which is a genus of Gram-negative bacteria known to be able to alter sex ratios, has been found at least in one polyctenid species, Hesperoctenes fumarius (Sakamoto et al., 2006), and is common in other bat ectoparasites (Morse et al., 2012;Wilkinson et al., 2016).
Nevertheless, there is a lack of evidence if they occur in a wide range of polyctenid species, and if they affect their reproduction. Future studies should address polyctenid sex ratios and their driving factors.
Prevalence of polyctenids shows a wide variation on their hosts, ranging from 2.4% to 85.2%. We currently have little understanding on what affects prevalence of these ectoparasites, although it is likely shaped by several factors, such as host availability, dispersal ability, seasonality, and population dynamics of each species.
Furthermore, data on potential host sex bias are not available or scarce; however, one study found equal infection between female and male hosts (Marshall, 1982b). Prevalence and infection pattern between host sexes need to be explored in future studies.

| Phylogenetic relationship of Polyctenidae
Previous phylogenetic trees involving Polyctenidae were based on morphological data (Schuh et al., 2009). Our genetic analysis placed Primicimicinae (Primicimex and Bucimex) to the base of the tree.
Polyctenid species cluster close to Primicimicinae, forming a separate clade at the base of Cimicidae. Based on these results, Polycteninae is a sister clade to Primicimicinae. Subfamily Cacodminae appears to be monophyletic, which has been shown before (Balvín et al., 2015;Hornok et al., 2021;Ossa et al., 2019;Roth et al., 2019). Subfamily Cimicinae also shows monophyly, with two separated clusters for the genus Cimex encompassing the genus Paracimex, which supports previous findings (Balvín et al., 2015;Roth et al., 2019).

| Potential as vectors
Polyctenidae have not been identified as vectors of any pathogens. However, they may have a potential role in disease transmission. Closely related bat bug species belonging to family Cimicidae are competent or suspected vectors of several pathogens, such as Trypanosoma, Bartonella, and Kaeng Khoi virus (Gardner & Molyneux, 1988;Reeves et al., 2005;Salazar et al., 2015;Van Den Berghe et al., 1963;Williams et al., 1976). The vector of Nycteria (Haemosporidia) parasites, which have been shown to infect, e.g., Rhinolophidae and Nycteridae species (Schaer et al., 2015), is not known and as some polyctenids parasitize these families, it is possible that they play a vectorial role in Nycteria transmission.
F I G U R E 2 Bayesian tree of family Cimicidae (including all six subfamilies) and Polyctenidae (including two species, one subfamily) based on concatenated sequences of the cytochrome c oxidase subunit 1 (COI), 16S, and 18S rRNA genes. GenBank accession numbers for each species are indicated in Table 3. Scale bar indicates the number of substitutions per site. Main host groups are indicated for each subfamily (i.e., birds, bats, and and/or humans). Formal analysis (equal); methodology (equal); writing -review and editing (equal). Nóra Takács

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available in GenBank at ncbi.nlm.nih.gov/genbank/, reference number ON157489 -ON182061. Philippe Christe https://orcid.org/0000-0002-8605-7002